Diagnostic tests and assays in the research field which are based on nucleic acid analysis are of still increasing importance. Since on the one hand, the nucleic acids are often present in very small concentrations and, on the other hand, they are often found in the presence of many other solid and dissolved substances, e.g., after lysis of cells or in sample material from food, they are difficult to isolate or to measure, in particular in biospecific assays which allow the detection of specific analytes. Therefore, in the majority of cases, these microbiological tests comprise at least one amplification step of the characteristic DNA molecules to be detected. A well-known assay which entails the selective binding of two oligonucleotide primers is the polymerase chain reaction (PCR) described in U.S. Pat. No. 4,683,195. This method allows the selective amplification of a specific nucleic acid region to detectable levels by a thermostable polymerase in the presence of deoxynucleotide triphosphates in several cycles. The PCR technology is a very sensitive technology with respect to both the required amount and the purity of the employed sample material.
Other possible amplification reactions are the ligase chain reaction (LCR, Wu, D., Y., and Wallace, R., B., Genomics 4 (1989) 560-569 and Barany, F., Proc. Natl. Acad. Sci. USA 88 (1991) 189-193); polymerase ligase chain reaction (Barany, F., PCR Methods and Appl. 1 (1991) 5-16); gap-LCR (PCT Patent Publication No. WO 90/01069); repair chain reaction (EP 0 439 182), 3SR (Kwoh, D., Y., et al., Proc. Natl. Acad. Sci. USA 86 (1989) 1173-1177; Guatelli, J., C., et al., Proc. Natl. Acad. Sci. USA 87 (1990) 1874-1878; PCT Patent Publication No. WO 92/0880A), and NASBA (U.S. Pat. No. 5,130,238). Further, there are strand displacement amplification (SDA), transcription mediated amplification (TMA), and Qβ amplification (for a review see e.g., Whelen, A., C. and Persing, D., H., Annu. Rev. Microbiol. 50 (1996) 349-373; Abramson, R., D. and Myers, T., W., Current Opinion in Biotechnology 4 (1993) 41-47).
As nucleic acids are only present within the cells of prokaryotic and eukaryotic organisms the cell wall has to be lysed prior to nucleic acid isolation. Concomitantly with the release of the nucleic acid from the cells, all other cellular components are also liberated. This includes proteins, salts, secondary metabolites as well as degradating enzyme, as e.g., proteases and nucleases. These enzymes start to degrade their target immediately. Thus the activity of these degrading enzymes has to be suppressed. This can be achieved by the addition of organic solvents or denaturating agents to the lysis solution. An alternative is the addition of protease and/or nuclease inhibitors.
In order to isolate nucleic acids from sample material there are several methods for the extraction of nucleic acids such as sequence-dependent or biospecific methods (e.g., affinity chromatography, hybridisation to immobilised probes) and sequence-independent or physico-chemical methods. Among the latter, well known to the art are liquid-liquid extraction with, e.g., phenol-chloroform, precipitation with an organic solvent such as ethanol, extraction with filter paper, extraction with micelle-forming agents such as cetyl-trimethyl-ammonium-bromide, interaction with immobilised, intercalating dyes such as acridine derivatives, as well as adsorption under chaotropic conditions to solid phases such as silica gel or diatomic earths, and adsorption to magnetic particles coated with, e.g., glass or magnetic organo silane particles.
Frequently, cationic surfaces are used to isolate nucleic acids. Such surfaces may be used to adsorb charged DNA molecules, whereby, e.g., EP 0 281 390 describes a polycationic support for nucleic acid isolation, WO 01/94573 charged membranes or WO 00/69872 a pH dependent ion exchange matrix. WO 02/48164 discloses polymers with switchable charge on solid supports for reversible binding of DNA. Similar to cationic surfaces, polycationic entities have certain DNA-binding affinity, too. Stewart, K., D., et al., J. Phys. Org. Chem. 5 (1992) 461-466 reports an increasing affinity of polyamines in solution for binding to DNA with increasing cationic charge. Doré, K., et al, J. Am. Chem. Soc. 126 (2004) 4240-4244 describes the selectivity of cationic compounds between double-stranded and single-stranded nucleic acids.
Another approach, normally applied to the separation and isolation of, e.g., DNA from complex biological fluids, is the use of nucleic acid binding materials. For example, the most prominent example of DNA binding material are glass surfaces due to their ability to reversibly bind DNA in the presence of chaotropic reagents and/or alcoholic additives (Vogelstein, B., and Gillespie, D., Proc. Natl. Acad. Sci. USA 76 (1979) 615-619). Such binding is assumed to be effected by oxidic surfaces (“X—OH”) interacting with phosphate groups of the nucleic acids.
A common method for the isolation of nucleic acids was published 1987 by Chomczynski, P., and Sacchi, N., Anal. Biochem. 162 (1987) 156-159. This method exploits the different solubilities of proteins and nucleic acids for an extractive separation protocol with an acidic guanidinium thiocyanate—phenol/chloroform mixture.
Boom, R., et al., J. Clin. Microbiol. 28 (1990) 495-503 describes a small scale protocol for the purification of DNA and RNA from sample material. The method is based on the lysing and nuclease-inactivating properties of a chaotropic agent in the presence of an EDTA/detergent mixture and the nucleic acid-binding properties of silica particles.
Lithium salts of nucleic acids are known to have a reduced solubility in aqueous solutions. In the European Patent Application EP 0 818 461 a method for the isolation of ribonucleic acid with an acidic solution containing a lithium salt and a chaotropic agent as well as an nucleic acid-binding partner such as silica particles is described.
In the U.S. Pat. No. 5,808,041 a composition for isolating nucleic acids from cells is described. The compositions are mixtures of silica gel and glass particles combined with chaotropic salts.
In WO 99/61603 a method for separating and/or isolating circular nucleic acids under alkaline conditions at a pH>8 with a solid matrix consisting essentially of a silica material in presence of at least one chaotropic substance is described.
US patent application 2004/0121336 describes a method of binding a predetermined amount of a nucleic acid to a multiplicity of solid substrate binding units. A method for gently lysing and solubilizing cells is described in US patent application 2004/0180445.
In view of certain disadvantages of the state of the art, it is the objective of the current invention to provide an alternative method for the isolation and purification of nucleic acid molecules from complex sample material. A particular object of the invention is to provide alternative compounds to promote the adsorption of a nucleic acid to a solid substrate.